Due to their role in regulating T cell activation, costimulatory molecules and their downstream pathways have been targeted in the treatment of diseases driven by T cell activation, such as graft versus host disease (GVHD). However, blockade of known costimulatory molecules improves, but not abrogate disease pathogenesis, suggesting contributions from additional, yet to be identified costimulatory molecules. We identified a novel function for the cell surface receptor LRRC8A as a co-stimulatory receptor essential for T cell activation by non-professional antigen-presenting cells (APCs). This proposal aims to determine the mechanistic basis for LRRC8A costimulation and its potential as a novel therapeutic target for preventing acute GVHD, the major cause of post-transplant morbidity and mortality. We show that LRRC8A is a receptor on the surface of T cells with an extracellular C-terminus. LRRC8A co-localizes with the T cell receptor (TCR)/CD3 complex after anti-CD3 stimulation and enhances TCR-driven T cell proliferation and cytokine secretion. LRRC8A ligation activates proximal signaling molecules shared by the TCR, specifically a Src kinase-Zap-70-PI3K pathway necessary for activation of AKT, a serine/threonine kinase important for cell survival. Soluble LRRC8A binds to fibroblasts and keratinocytes, but not dendritic cells or B cells, suggesting that non-hematopoietic APCs express a ligand for LRRC8A. Notably, we identified a specific role for LRRC8A in T cell activation: blocking the LRRC8A receptor-ligand interaction inhibits T cell proliferation to antigen presented by non-hematopoietic APCs in vitro as well as tissue inflammation induced by contact hypersensitivity to hapten in vivo. These findings suggest that LRRC8A may be a therapeutic target for diseases dependent on T cell activation induced by non-hematopoietic APCs. This is particularly relevant to GVHD since recipient non- hematopoietic APCs are necessary and sufficient for initiating CD4+ T cell-driven acute GVHD in models of MHC-identical, T cell-replete hematopoietic stem cell transplantation, which are analogous to the majority of human MHC-matched allogeneic transplants. However, a detailed, mechanistic understanding of how LRRC8A exerts its costimulatory effect is needed to determine its therapeutic applications. Our central hypothesis is that LRRC8A activates signaling pathways in T cells that overlap with those activated by CD3 ligation, thereby delivering a costimulatory signal essential for acute GVHD induced by antigen-presenting non-hematopoietic cells. We have developed unique reagents ideal for this study, including conventional and conditional LRRC8A-deficient mice, anti-LRRC8A antibodies, and soluble LRRC8A fusion proteins. The study will be executed with the mentorship of three leaders in the fields of immunobiology, T cell signaling, and GVHD. Using this infrastructure, we will test the following specific aims: Specific Aim 1: Dissect the molecular and cellular mechanisms underlying LRRC8A-mediated costimulation Specific Aim 2: Evaluate the LRRC8A receptor-ligand interaction as a therapeutic target for acute GVHD The applicant, Dr. Janet Chou, is an instructor in the Division of Immunology, Boston Children's Hospital. The 5-year plan builds on her prior background in T cell biology and clinical immunology. To achieve these specific aims, she has proposed a career development plan with two educational objectives: (1) master experimental methods for dissecting T costimulatory pathways and (2) acquire expertise in murine models of graft versus host disease. The primary mentor for this project, Dr. Raif Geha, is an internationally-recognized investigator in mechanisms of host immunity and primary immunodeficiency. The co-mentors, Drs. Cornelis Terhorst and Jerome Ritz, are experts in the fields of T cell costimulatory molecules and GVHD, respectively. The study will be executed within the Division of Immunology at Boston Children's Hospital, which has a robust infrastructure for basic and translational research and a superb history of mentoring physician-scientists. This K08 award will enable Dr. Chou establish the foundation for her long-term career goal: creating a translational immunology research program that develops rational targeted approaches for immunomodulation through the study of molecular mechanisms underlying host immunity.